1. Regulated Osteopontin Expression by Dendritic Cells Decisively Affects their Migratory Capacity 
Guido Schulz, Andreas C. Renkl, Anne Seier, L. Liaw, Johannes M. Weiss 
Journal of Investigative Dermatology 
Volume 128, Issue 10, Pages (October 2008)
DOI: /jid
Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Upregulation of constitutive OPN secretion by TNF-α and IL-1α correlates with the migratory capacity of DCs. DCs were generated from bone marrow of mice (C57BL/6) in the presence of GM-CSF (40ngml−1; PromoCell, Heidelberg, Germany) and IL-4 (10ngml−1; Cell Concepts, Umkirch, Germany) for 5 or 6 days as described previously (Weiss et al., 2001). During DC differentiation, supernatants and cells were obtained daily. In all the figures, data representative of at least four independent experiments is shown. (a) OPN concentration in supernatants obtained on the indicated days of culture was measured by OPN specific ELISA (IBL-Japan, Gunma, Japan) according to the manufacturer's instructions. (b) OPN quantitative real-time PCR of cells harvested on the indicated days was performed with a LightCycler (Roche Diagnostics, Mannheim, Germany) using the following primers: OPN (108-bp product): sense, 5′-GGTGATAGCTTGGCTTATGGACTG-3′; antisense, 5′-GCTCTTCATGTGAGAGGTGAGGTC-3′; glyceraldehyde-3-phosphate dehydrogenase (147-bp product): sense, 5′-TGGCCTTCCGTGTTCCTACC-3′; antisense, 5′-GGTCCTCAGTGTAGCCCAAGATG-3′. Thermocycling conditions were as follows: hold at 95°C for 15minutes, 40 cycles of 95°C for 15seconds, 60°C for 20seconds, and 72°C for 10seconds followed by melting-curve analysis. The relative expression of target gene in different samples was normalized to the endogenous glyceraldehyde-3-phosphate dehydrogenase and was calculated with the 2−ΔΔCt method (Livak et al., 2001). (c) For further DC enrichment, CD11c+ cells were positively selected by magnetic cell sorting (Miltenyi, Bergisch Gladbach, Germany) and quantitative real-time PCR was performed for OPN as described above. Unseparated bone marrow DC culture cells were used as control. (d) DCs were harvested from bone marrow cultures on day 5, washed, replated (1 Mio. cells per well) and stimulated with LPS (serotype 0111:B4; Sigma-Aldrich, Hamburg, Germany) at the indicated concentrations. Viability was determined by propidium iodide staining by fluorescence-activated cell sorting and did not significantly differ for unstimulated controls and the indicated LPS concentrations (93–95% viability, after 48hours of culture). (e) DCs were harvested from bone marrow cultures on day 5, washed and cultured with or without GM-CSF (20ngml−1) for 48hours without additional cytokines, GM-CSF or as indicated additional IL-4 (5ngml−1), TNF-α (100Uml−1; Cell Concepts), IL-1α (500pgml−1; Cell Concepts) or LPS (1μgml−1, serotype 0111:B4; Sigma-Aldrich) at 0.5 Mio. Cells per well. Supernatants were obtained after 48hours of stimulation to detect OPN concentration by ELISA. To investigate the effect of OPN production on their migratory capacity, DCs were compared in their migratory function towards TNF-α in modified Boyden chamber assays. Cells that had migrated to the filter bottom were quantified counting four ocular grids (× 40). Results are expressed as mean number of migrated cells per mm−2 ±SD of six chambers. *Statistically significant compared with GM-CSF control (t-test P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
OPN-deficient DCs are impaired in their capacity to migrate in vitro and to enter skin draining lymph nodes in vivo. DCs were generated from bone marrow of OPN-deficient C57BL/6 mice (-/-) (tenth generation of backcrossing) and wild-type littermates (+/+) for 6 days and used in Boyden chamber migration assays to migrate toward (a) OPN (2.5μgml−1, recombinant OPN), (a) TNF-α (50 and 250Uml−1), or (b) CCL19, R&D Systems, Wiesbaden, Germany (100 or 300ngml−1). Hank's balanced salt solution medium was used as control. Cells that had migrated to the filter bottom were quantified as described above. *Statistically significant compared with corresponding +/+ DCs (t-test P<0.001). Representative data of four independent experiments is shown. (c) In vivo DC migration assays were performed as described previously (Lappin et al., 1999). All animal protocols were approved by the local Committee of Animal Research. DCs were generated from OPN-null (-/-) C57BL/6 mice or wild-type littermates (+/+) and labeled with PKH2-GL (Sigma Immunochemicals, Hamburg, Germany) and injected at 250,000 DCs in 30μl of phosphate-buffered saline into each quadrant of the abdominal skin of wild-type mice. Twelve wild-type mice were injected with OPN wild-type DCs and 14 mice with OPN-null DCs. Inguinal and axillary lymph nodes were obtained after 48hours, mechanically disaggregated, pooled, stained with phycoerythrin-labeled antibody against mouse CD11c (clone HL3, hamster IgG; Pharmingen, Heidelberg, Germany). CD11c, PKH2-GL double positive cells were quantified by a FACS. Using Mann–Whitney rank-sum test the difference between the two groups is statistically significant (P=0.042).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions